High molecular and low molecular fractions of von willebrand factor

ABSTRACT

The invention provides high and low molecular weight fraction of von Willebrand Factor (vWF), which can be obtained by absorbing vWF to a heparin affinity support followed by eluting the vWF at differing salt concentrations.

This application is a divisional, of application Ser. No. 08/538,889,filed Oct. 4, 1995.

DESCRIPTION

The invention relates to a method for fractionation of von WillebrandFactor into a high molecular and low molecular fraction.

Further, the invention relates to a low molecular fraction of vonWillebrand Factor (vWF) molecules, a high molecular fraction of vonWillebrand Factor molecules as well as a mixture of vWF molecules of thelow molecular and high molecular fraction.

Direct and indirect functions are assigned to von Willebrand Factor innormally proceeding blood coagulation. It binds to Factor VIII in acomplex. This complex serves to stabilize Factor VIII. This stabilizedFactor VIII then has essential cofactor function in the activation ofFactor X. Additionally, von Willebrand Factor directly influences bloodcoagulation by mediating platelet aggregation to injured vessels.

In plasma, vWF circulates in a concentration of 5-10 mg/l in the form ofa non-covalent complex with Factor VIII. vWF is a glycoprotein which isformed in different cells of the human body and is later released intothe circulation. Moreover, starting from a polypeptide chain with amolecular weight of about 220,000 (vWF monomer), a vWF dimer (primarydimer) with a molecular weight of about 550,000 is made in cells by theformation of several sulfur bridges. Then, further polymers of vWF withincreasing molecular weights up to about 20 million are produced fromthe vWF dimers by association.

There are several clinical pictures which are traceable to under- oroverproduction of von Willebrand Factor. Thus, for example, anoverproduction of vWF leads to an increased tendency towards thromboses,whereas an undersupply of vWF results in an increased bleeding tendencyor prolonged bleeding time.

von Willebrand Syndrome can manifest itself in several forms. All formsare distinguished by a prolonged bleeding time which is based on eitheran absolute absence of a functional vWF or an abnormal spectrum in themultimer composition of vWF. Forms of von Willebrand disease in whichmultimer formation is reduced as well as forms in which low molecularvWF molecules are barely present are diagnosed thereby. Although otherforms demonstrate high and low molecular vWF molecules, theirconcentration and/or their ratio to each other is drastically decreasedand/or altered compared to a healthy person.

The lack of vWF can also cause hemophilia A because vWF, as mentionedabove, is an essential component of functional Factor VIII. In thesecases, the half-life of Factor VIII is decreased in such a manner thatit can not fulfill its special functions in the blood coagulationcascade.

All forms of von Willebrand Syndrome as well as the form of hemophiliatraceable to the lack of vWF were treated up to now by replacement ofthe missing vWF through intravenous infusions with concentrates of bloodplasma which contain either vWF-Factor VIII complex or enriched vWF.Although in one respect the administration of Factor VIII is notnecessary in both disease cases, the preparative separation of vWF fromFactor VIII is technically very difficult to impossible.

In order to establish the exact function of high molecular vWF moleculeson the one hand and low molecular vWF molecules on the other hand, wayshave been looked for to isolate these fractions in enriched form.Wagner, D. D. et al (J. Cell Biol. 101: 112, 1985) prevent multimerformation by addition of monensin in vitro and conclude from differentexperiments that the low molecular form of vWF is not functional. On theother hand, Senogles, S. E. et al (J. Biol. Chem. 258: 12327, 1983) findno functional difference in ristocitin mediated platelet aggregation inhigh molecular and low molecular forms of vWF. They obtain low molecularforms by reduction of the sulfur bridges of high molecular vWFmolecules. Aihara, M. et al (Tohoku J. Exp. Med. 153: 169, 1985) observea lower binding capacity of vWF molecules from patients with vonWillebrand Syndrome of the type IIa to collagen in affinitychromatography. This type of von Willebrand Syndrome is characterized byabsence of the high molecular molecules of vWF.

In the literature, there are numerous methods which describe ananalytical separation of high molecular and low molecular forms of vonWillebrand Factor such that statements can be made as to thequantitative proportion of both forms. As an example, a publication fromBaillod et al in Thrombosis Res. 66: 745, 1992 should be mentioned here.However, a preparative method for the separation of low molecular andhigh molecular forms has not been described to date.

SUMMARY OF THE INVENTION

The object of the present invention is to develop a preparative methodfor the fractionation of von Willebrand Factor (vWF) into a highmolecular and low molecular fraction of von Willebrand Factor.

This object is solved by a method for the separation of vWF, especiallyof recombinant von Willebrand Factor, into high molecular vWF and lowmolecular vWF which is characterized in that vWF is bound to an affinitysupport and then eluted by different salt concentrations.

Preferred embodiments include separation methods where the vWF to beseparated is present in a plasma fraction enriched with vWF.Alternatively, the vWF to be separated is recombinant vWF which ispresent in a recombinant vWF concentrate from cell free culturesupernatants of transformed cells.

The purified vWF can be fractionated into high molecular vWF and lowmolecular vWF. The separation of vWF into high molecular vWF and lowmolecular vWF preferably is performed in a Ca²⁺ -free buffer system. Thelow molecular vWF can be eluted at a lower salt concentration than highmolecular vWF.

For example, vWF can be bound to the affinity support at a saltconcentration <150 mM, low molecular aggregates of vWF are eluted at asalt concentration between 150 and 250 mM, preferably 160 mM, andthereafter, high molecular aggregates of vWF are eluted at a saltconcentration of >250 mM, preferably ≦270 mM. Soluble mono- and divalentsalts can be used as salts. A preferred salt is NaCl.

The affinity support is preferably a support with Heparin bound thereto,preferably using AF-heparin Toyopearl® (Tosohaas), HeparinEMD-Fraktogel® (Merck) or Heparin Sepharose Fast Flow®. The buffersolutions disclosed herein can comprise buffer substances and optionallysalt, and can be used as a buffer system for affinity chromatography.For example, a buffer solution comprising Tris/HCl buffer, phosphatebuffer or citrate buffer, and optionally sodium chloride, can used as abuffer system. Preferably, the affinity chromatography is carried out ina pH range of 6.0 to 8.5, more preferably at a pH value of 7.4.

In a particularly preferred embodiment of the present invention, theseparation is performed in a Ca²⁺ -free buffer system. In this manner,high molecular vWF fractions or high molecular rvWF fractions whichpossess a particularly high physiological activity can be obtained withgood yield.

Further subject matter of the present invention is a low molecularfraction of vWF molecules which comprises dimers and tetramers, wherebythe dimers and tetramers consist of identical vWF subunits.Additionally, the invention relates to a low molecular fraction of vWFmolecules which is obtainable by separation of vWF into high molecularvWF and low molecular vWF with an affinity support through elution atdifferent salt concentrations. Various parameters for undertaking suchseparation are described above.

Preferred embodiments include low molecular fractions of vWF moleculeswhich comprises at least 83% dimers and maximally 16% tetramers andmaximally 1% higher polymers. The low molecular fraction of vWFmolecules can be free from platelet aggregating action. The lowmolecular fraction of vWF molecules can bind to Factor VIII, contributeto the stabilization of Factor VIII and positively influences thestorage stability of Factor VIII. Preferably, the low molecular fractionof vWF molecules comprises plasmic vWF and/or recombinant vWF.

The present invention relates to a high molecular fraction of vWFmolecules which has an at least 50%, preferably 60%, improved activityper μg protein in platelet aggregation compared with the physiologicalmixture of high molecular and low molecular vWF molecules. Additionally,the invention relates to a high molecular fraction of vWF moleculesobtainable by separation of vWF into high molecular vWF and lowmolecular vWF with an affinity support through elution at different saltconcentrations. Various parameters for undertaking such separation aredescribed above. According to the invention, the activity per μg proteincan be even further increased.

Preferred embodiments of the high molecular fraction are plasmic vWFand/or recombinant vWF.

The invention further relates to a mixture of the low molecular fractionof vWF molecules described above and the high molecular fraction of vWFmolecules described above in any mixture ratio.

A preferred mixture is a mixture ratio in which the portion of lowmolecular vWF is lower than 35% or higher than 45%.

The invention also relates to the use of the low molecular fraction ofvWF molecules or the high molecular fraction of vWF molecules or amixture thereof in any mixture ratio for the treatment of hemophilia Aor different forms of von Willebrand Disease.

In view of the prior art, it was not predictable that the fractionsobtainable according to a preparative separation method and/or theirmixtures are suitable for the treatment of the diseases mentioned.

Subject matter of the invention is also a pharmaceutical compositionwhich comprises the low molecular fraction of vWF molecules or the highmolecular fraction of vWF molecules or a mixture thereof in any mixtureratio in a physiologically acceptable carrier.

A preferred composition comprises Factor VIII or functional deletionmutant of Factor VIII, wherein the vWF molecules of the high molecularfraction or the low molecular fraction or the mixture thereof stabilizesFactor VIII or functional deletion mutations of Factor VIII.

The method according to the invention is suitable in the same manner forplasmic as well as recombinant vWF (rvWF). The starting material for theseparation of the high and low molecular fractions is either a vWFenriched plasma fraction or a cell-free culture medium afterfermentation of animal cells from which recombinant vWF was isolated andpre-purified.

The vWF used for the separation can be pre-purified with the aid of anyknown method.

According to the method of the invention for the separation of vWF, andespecially rvWF, into high molecular and low molecular fractions, vWF isbound to an affinity support and then eluted at different saltconcentrations.

To obtain particularly high yields, the separation is carried out in aCa²⁺ -free buffer system.

The low molecular vWF fractions can be eluted at a lower saltconcentration than the high molecular vWF fractions.

Soluble mono- and divalent salts are useable for the elution.Preferably, NaCl is used. Calcium salts are not suitable for theelution.

In a preferred embodiment, vWF is bound to the affinity support at asalt concentration of <150 mM. At a salt concentration between 150 and250 mM, and especially 160 mM, the low molecular aggregates of vWF arethen eluted, and thereafter, at a salt concentration of >250 mM, andespecially at >270 mM, the high molecular aggregates are eluted.

NaCl is preferred as a salt. Calcium salts are not suitable.

The method according to the invention is preferably carried out on aheparin affinity chromatography column. Any support on which heparin canbe bound can be used for the affinity chromatography. For example,AF-HEPARARIN-TOYOPEARL® (a synthetic, large-pored, hydrophilic polymerbased on methacrylate (Tosohaas), HEPARIN EMD-FRAKTOGEL® (a synthetic,hydrophilic polymer based on ethylene glycol, methacrylate anddimethylacrylate) (Merck) or SEPHAROSE FAST FLOW® (containing naturaldextran and/or agarose derivatives) (Pharmacia) have demonstratedthemselves as well suited.

The affinity chromatography is preferably performed in a pH range of 6.0to 8.5, and especially at a pH value of 7.4.

In the method according to the invention, a buffer solution comprisingbuffer substances, especially Tris/HCl buffer, phosphate buffer orcitrate buffer, and optionally salt, which is preferably free fromstabilizers, amino acids and other additives is used as a buffer system.It was shown that a particularly good separation of low molecular andhigh molecular vWF protein aggregates can be achieved with a vWFsolution treated with EDTA in a Ca²⁺ -free buffer system. Therefore, inthis manner, high molecular vWF fractions or high molecular rvWFfractions which have a particularly high physiological activity can alsobe obtained with good yield.

In the method according to the invention, a recombinant vWF concentratefrom cell-free culture supernatants of transformed cells is preferablyemployed.

According to the method of the invention for the separation of vWF intohigh molecular and low molecular fractions, low molecular and highmolecular vWF can be obtained in an efficient and simple manner.Therefore, according to this separation method, the particularlyphysiologically active high molecular or low molecular fractions of vWFwhich are therewith exceptionally suitable for the treatment ofhemophilia A and different forms of von Willebrand Disease can beproduced with good yield.

In a preferred embodiment according to the invention, purified vWF isfractionated into high molecular vWF and low molecular vWF.

The vWF employed for the separation can be pre-purified with the aid ofany known method.

Particularly preferred is the purified von Willebrand Factor obtainableaccording to a method which comprises the steps of chromatographicallypurifying plasmic von Willebrand Factor by an anion exchangechromatography on an anion exchanger of the quaternary amino type and anaffinity chromatography on immobilized heparin in a buffer solutioncomprising buffer substances and optionally salt.

In a further preferred embodiment, the purified von Willebrand Factor isobtainable according to a method which comprises the steps ofchromatographically purifying recombinant von Willebrand Factor by ananion exchange chromatography on an anion exchanger of the quaternaryamino type and an affinity chromatography on immobilized heparin in abuffer solution comprising buffer substances and optionally sodiumchloride.

Particularly preferred is the purified von Willebrand Factor obtainableaccording to a method in which a rvWF concentrate is purified fromcell-free culture supernatants of transformed cells.

In the purification of vWF, a buffer system free from stabilizers, aminoacids and other additives is particularly preferred as a buffersolution.

The anion exchange chromatography and/or the affinity chromatography ispreferably carried out in a pH range of 6.0 to 8.5, and more preferablyat a pH value of 7.4.

The von Willebrand Factor bound on the anion exchanger in anion exchangechromatography and on immobilized heparin in affinity chromatography canbe eluted by increasing the salt concentration.

The quaternary anion exchanger of a Fraktogel with tentacle structure ispreferred, especially an EMD-TMAE-Fraktogel.

In the purification method, the vWF on the anion exchanger is preferablybound at a salt concentration of ≦270 mM NaCl and eluted at a saltconcentration of >270 mM NaCl, and preferably >280 mM NaCl.

In the purification of vWF, affinity chromatography is preferablycarried out on a support with heparin bound thereto, whereby preferablyAF-HEPARIN-TOYOPEARL® (Tosohaas), HEPARIN EMD-FRAKTOGEL® (Merck) andHEPARIN SAPHAROSE FAST FLOW® are equally suitable.

In a preferred embodiment, the purified von Willebrand Factor isobtainable according to a method in which the vWF, pre-purified in anionexchange chromatography, binds to the immobilized heparin at a saltconcentration of <150 mM NaCl and is eluted at a salt concentrationof >150 mM NaCl, preferably at 200 to 300 mM NaCl, more preferably 160to 270 mM NaCl.

It is advantageous to employ previously purified concentrates as thestarting material for the separation of high molecular and low molecularvWF. Additionally, the chromatographic separation method results in afurther purification effect.

The high molecular and low molecular fractions obtained according to theseparation method of the invention as well as mixtures of both fractionsin any mixture ratio are physiologically active and can be employed fortherapeutic purposes.

The low molecular fraction of vWF molecules is characterized in that itpredominantly comprises dimers and tetramers which consist of identicalvWF subunits.

The portion of dimers and tetramers in the low molecular fraction ishigher than in the physiological composition of vWF.

It was surprisingly found that low molecular fractions of recombinantvWF consist of identical vWF subunits, whereas fractions of plasmic vWFconsist of a mixture of dimers and tetramers with different subunits.

The low molecular fraction of vWF molecules is obtainable according to amethod described above.

In a preferred embodiment, the low molecular fraction of vWF moleculescomprises at least 83% dimers and maximally 16% tetramers and maximally1% higher polymers. The low molecular fraction is free from plateletaggregating action, binds Factor VIII, contributes to the stabilizationof Factor VIII and positively influences the storage stability of FactorVIII.

The low molecular fraction of vWF molecules preferably consists ofrecombinant vWF.

The high molecular fraction of vWF molecules has an at least 50%,preferably 60%, improved activity per μg protein in platelet aggregationas compared with the physiological mixture of high molecular and lowmolecular vWF molecules. The high molecular fraction of vWF molecules isobtainable according to a method described above.

It was surprisingly found that the high molecular fraction ofrecombinant vWF comprises multimers which consist of identical vWFsubunits, whereas fractions of plasmic vWF consist of a mixture ofmultimers with different subunits.

The high molecular fraction of vWF molecules preferably consists ofrecombinant vWF.

Surprisingly, it was found that the low molecular as well as the highmolecular fraction of recombinant vWF possess a potentially higherbinding capacity to Factor VIII in comparison to corresponding fractionsof plasmic vWF. Hence, the fractions of recombinant vWF bind Factor VIIImore efficiently than the plasmic fractions.

The different use of the high molecular and/or low molecular fractionsresults from their physiological activity. The possibility of theproduction of purposive mixtures of both fractions permits the treatmentof special medical indications.

Hence, further subject matter of the present invention is the use of thelow molecular fraction of vWF molecules or the high molecular fractionof vWF molecules or a mixture thereof for the treatment of hemophilia Aor different forms of von Willebrand Disease.

It is especially pointed out that when using recombinant vWF in theseparation method according to the invention, the end products (highmolecular and low molecular fractions as well as mixtures of bothfractions in any mixture ratio) are free from plasma proteins and freefrom Factor VIII as well as free from pathogenic viruses. In theproduction of pharmaceutical products, as they are mentioned above, thiscan be of great advantage for certain medical indications.

Subject matter of the present invention is also a pharmaceuticalcomposition which comprises the low molecular fraction of vWF moleculesor the high molecular fraction of vWF molecules or a mixture thereof inany mixture ratio in a physiologically acceptable carrier.

Preferably, the pharmaceutical composition comprises Factor VIII orfunctional deletion mutation(s) of Factor VIII, whereby the vWFmolecules of the high molecular fraction or the low molecular fractionor the mixture thereof stabilize Factor VIII or functional deletionmutation(s) of Factor VIII.

For the production of pharmaceutical preparations, the respectivefractions or their mixtures are preferably concentrated and theconcentrate is further processed.

The production of the pharmaceutical compositions can occur in a knownand customary manner. Preferably, the products (low molecular and highmolecular fractions as well as any possible mixture thereof) or theconcentrates comprising these can be mixed with a suitablephysiologically acceptable carrier. Preferably, a physiological sodiumchloride solution serves as a carrier.

The pharmaceutical compositions can be present in an administration formcustomary and usual for the treatment of hemophilia A and differentforms of von Willebrand Disease; preferably they are present in the formof a preparation suitable for infusion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the multimer spectrum of individually obtained vWF froman SDS-agarose gel electrophoresis. FIG. 2 depicts a densitometricanalysis of the distribution of multimers of a recombinant vWF fractionobtained by elution with 160 mM NaCl.

FIG. 3 depicts a densitometric analysis of the distribution of multimersof a recombinant vWF fraction obtained by elution with 270 mM NaCl.

FIG. 4 depicts the binding of Factor VIII to von Willebrand Factor as afunction of the available amount of Factor VIII.

FIG. 5 depicts a denaturing electrophoretic analysis of the purifiedrvWF.

FIG. 6 depicts a sensogram of the attachment of r-Factor VIII to r-vWF,which shows the kinetics of the interaction between these factors.

FIGS. 7a, 7b and 7c depict an electrophoretic analysis of the fractionswith different degrees of polymerization after separation of vWFmultimers of p-vWF, K-vWF and r-vWF.

FIG. 8 depicts an electrophoretic analysis of p-vWF and r-vWF afteraffinity chromatography and elution with 280 NaCl.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following Examples, the invention is more closely illustratedwithout restricting the invention to the Examples.

In Example 1, the separation according to the invention of highmolecular and low molecular vWF polymers by means of heparin affinitychromatography is shown.

EXAMPLE 1

Separation of high and low molecular forms of von Willebrand Factor

Recombinant von Willebrand Factor (rvWF) in 20 mM Tris/HCl buffer (Trisbuffer), pH 7.4, was applied to a glass column which was filled withAF-HEPARIN-TOYOPEARL® (Tosohaas) with a flow speed of 1 ml/min/cm². Thecolumn was first washed with Tris buffer in order to removenon-specifically bound proteins. Then, the column was eluted with 160 mMNaCl in Tris buffer and subsequently with 270 mM NaCl in Tris buffer.During the chromatography, the protein absorption was followed in acustomary manner at 280 nm. After the chromatography, the proteinconcentration was determined by means of the Bradford method (M.Bradford, Anal. Biochem. 72: 248-254, 1976). The content of rvWF wasdetermined by means of a commercial ELISA system (Boehringer Mannheim).The distribution of multimer structures of recombinant von WillebrandFactor in the individual purification fractions was examined by SDSagarose gel electrophoresis in the customary manner and quantitativelyanalyzed by densitometry. The capacity of ristocetin mediated bloodplatelet aggregation was examined by means of a commercial test system(von Willebrand Reagent, Behring Werke).

FIG. 1 shows the multimer spectrum of the individually obtainedfractions. At a salt concentration of 160 mM NaCl, rvWF with lowmolecular weight, predominantly of the primary diner type, was eluted.In contrast, rvWF with high molecular weight was eluted at a saltconcentration of 270 mM NaCl.

FIG. 2 shows a densitometric analysis of the distribution of multimersof recombinant von Willebrand Factor obtained at 160 mM NaCl. Thisanalysis demonstrated that the rvWF-polymer mixture obtained in thismanner comprised up to 84.4% of the primary diners (molecular weightabout 440,000) and up to 14.9% of the tetramers (molecular weight about880,000).

A densitometric analysis of the rvWF fraction shown in FIG. 3 which wasobtained by elution with 270 mM NaCl demonstrated that the rvWF-polymermixture obtained in this manner comprises a cascade of increasinglyhigher polymers.

The results of the chromatography and the first functional analysis ofrvWF fractions are compiled in Table 1. From the analysis it emergesthat nearly the entire rvWF was bound to the support. The rvWF-polymermixture eluted with 160 mM NaCl (low molecular) demonstrated no capacityfor agglutination of blood platelets. The fraction eluted at 270 mM NaCl(high molecular) possessed a high activity with respect to plateletaggregation. By the fractionation of the non-aggregating, low molecularrvWF forms, the specific activity with respect to the agglutination wasincreased by 69% compared to the starting material.

At the same time, the purity of the rvWF was substantially increased bychromatography of the rvWF on the affinity support described inExample 1. rvWF of the 160 mM NaCl fraction showed a double, and rvWF ofthe 270 mM NaCl fraction a 6-fold, higher purity in comparison to thestarting material.

                                      TABLE 1    __________________________________________________________________________              Volume                  Protein                       vWF:AG                            vWF:AG                                 μg vWF:AG/                                       Agglutination                                              Agglutination    Sample    ml  mg/ml                       μg/ml                            mg   μg Protein                                       mU/μg Protein                                              mU/μg vWF:AG    __________________________________________________________________________    rvWF Start              560 0.62 92   51   0.15  0.650  4.3    160 mM NaCl eluate              98  0.2  54   6    0.27  0      0    270 mM NaCl eluate              86  0.5  440  38   0.88  6.4    7.27    __________________________________________________________________________

EXAMPLE 2

Binding of recombinant von Willebrand Factor to Factor VIII

The following experiments were carried out in ELISA microtitrationplates which were coated with anti-vWF immunoglobulin (Asserachrom vWF,Boehringer Mannheim). 5 ng of vWF respectively was coupled to thereaction container by 1 hour incubation at room temperature with 200 μl50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.5% albumin, 0.1% Tween 20 (TBSbuffer) which contained 100 ng/ml vWF:AG. The following vWF samples wereused: human vWF (Diagnostika Stago), rvWF (starting material fromExample 1), low molecular rvWF(160 mM NaCl elution fraction from Example1), high molecular rvWF (270 mM NaCl elution fraction from Example 1).The microtitration plates were subsequently washed 3 times for 3 minuteswith TBS and subsequently each reaction container was covered with alayer of 25 μl recombinant Factor VIII (Miles) in TBS and incubated for1 hour at room temperature. The concentration of Factor VIII was variedin the range of 100 mU/ml to 7.12 mU/ml. Thereafter, the reactioncontainers were washed 3 times for 3 minutes each with 200 μl TBS.Subsequently, the amount of Factor VIII bound by the immobilized vWF wasdetermined by means of a commercial test system (Chromogenix CoatestVIII:C/4).

FIG. 4 shows the binding of Factor VIII to von Willebrand Factor as afunction of the available amount of Factor VIII. The results demonstratethat all examined vWF fractions bind Factor VIII in a concentrationdependent manner, whereby rvWF demonstrated an approximately identicalFactor VIII binding compared with human vWF. rvWF of the 270 mM NaClfraction from Example 1 demonstrated an increased binding of FactorVIII; rvWF from the 160 mM NaCl fraction, an insignificantly lowerbinding of Factor VIII.

EXAMPLE 3

Stabilization of recombinant Factor VIII by recombinant von WillebrandFactor

Factor VIII is physiologically bound in the human body by von WillebrandFactor and is stable in this form. As opposed to this, unbound FactorVIII is proteolytically inactivated in a few minutes.

Different fractions of recombinant von Willebrand Factor obtainedaccording to the method from Example 1 were added to transformed SK-Hepcells in cell culture which secrete Factor VIII. After 24 hours, theactivities of recombinant Factor VIII in the cell culture supernatantswere determined by means of a commercial test system (ChromogenixCoatest VIII:C/4). The results are compiled in Table 2. From the data,it is evident that a substantially higher concentration of Factor VIIIwas present after 24 hours by adding rvWF to the cells. The highmolecular vWF fraction as well as the low molecular vWF fraction bindand stabilize Factor VIII, whereby the high molecular fraction has abetter capacity for stabilizing Factor VIII than the low molecularfraction.

Additionally, these data demonstrate that recombinantly produced FactorVIII can also be stabilized by addition of von Willebrand Factor.

                  TABLE 2    ______________________________________    Sample         Factor VIII activity after 24 hours    15 μg/ml    U/ml    ______________________________________    control        0.5    rvWF start     1.8    rvWF-160 mM NaCl eluate                   0.8    rvWF-270 mM NaCl eluate                   2.4    ______________________________________

EXAMPLE 4

Stabilization of Factor VIII by recombinant von Willebrand Factor

Mixtures of Factor VIII and recombinant von Willebrand Factor from cellculture supernatants, as they were obtained in Example 3, were frozen at-20° C. and kept for 6 days at -20° C. Thereafter, the activity ofFactor VIII was determined again. Table 3 summarizes the results.

It is evident from the results that all tested vWF fractions increasethe storage stability of Factor VIII more that 2.5-fold. This alsoindicates a strong binding of vWF-Factor complexes with the lowmolecular fractions.

                  TABLE 3    ______________________________________    Sample          Factor VIII activity (mU/ml)    15 μg/ml     t = 0     t = 6 days                                       % loss    ______________________________________    control          500       150     70    rvWF start      1800      1300     28    rvWF-160 mM NaCl eluate                     800       600     25    rvWF-270 mM NaCl eluate                    2400      1800     25    ______________________________________

EXAMPLE 5

Purification of rvWF from culture supernatants by anion exchangechromatography

Recombinant vWF was isolated according to customary methods afterinfection of Vero cells (monkey kidney cells) with vaccinia virus incell culture. Vero/vaccinia expression systems and cell cultureconditions are described in detail in F.G. Falkner et al., Thrombosisand Haemostasis 68 (1992) 119-124, N. Barrett et al., AIDS Res. 5 (1989)159-171 and F. Dorner et al., AIDS Vaccine Research and Clinical Trials,Marcel Dekker, Inc., New York (1990). The expression of vWF occurred insynthetic DMEM standard medium (Dulbeccos minimal essential medium).

After the fermentation of the transformed cells, the culture medium wasfractionated and cells and cell fragments were removed bycentrifugation. Additional smaller components such as membrane fragmentsor bacteria were removed by filtration through a filter with a pore sizeof 0.4 μm.

770 ml of cell-free culture supernatant was filtered over a column (1.6cm×5 cm, filled with 10 ml of the anion exchanger EMD-TMAE-Fraktogel(Merck)) with a flow speed of 2 ml/cm² /min. The gel was previouslyequilibrated with 20 mM Tris/HCl buffer (pH 7.4). Subsequently, thecolumn was washed with 20 mM Tris/HCl buffer (pH 7.4). Foreign materialswere removed by washing the column with buffer containing 200 mM NaCi.The rvWF was then eluted from the support with 280 mM NaCl in 20 mMTris/HCl buffer (pH 7.4). Subsequently, any residual material presentwas eluted from the column with 1M NaCl. During chromatography, theprotein absorption was followed at 280 nm in the customary manner. Afterchromatography, the protein concentration was determined according tothe Bradford method (M. Bradford, Anal. Biochem. 72 (1976) 248-254). Thecontent of rvWF was determined by means of a commercial ELISA system(Boehringer Mannheim).

It was found that nearly the total rvWF was bound to the support. rvWFwas eluted from the anion exchanger by 0.28 M NaCl. The results of thepurification of rvWF on the anion exchanger are summarized in Table 4.

rvWF was enriched 6-fold by the purification described in this Example.

                  TABLE 4    ______________________________________             volume   Total Protein                                 rvWF   rvWF/    Sample   (ml)     (μg/ml) (μg/ml)                                        total protein    ______________________________________    cell-free             770      113        7.9    0.069    culture    supernatant    Elution with             95       147        0.0016 0.00001    200 mM NaCl    Elution with             75       168        61     0.36    280 mM NaCl    Elution with             50       196        6      0.03    1 M NaCl    ______________________________________

EXAMPLE 6

Purification of rvWF by affinity chromatography

rvWF obtained according to Example 5 was diluted with 20 mM Tris/HClbuffer (pH 7.4) to decrease the salt concentration (160 mM NaCl).Subsequently, the solution was filtered through a column (1.6 cm×5 cm,filled with 10 ml of AF heparin Toyopearl 650 (Tosohaas)) with a flowspeed of 1 ml/cm² /min. The column was previously equilibrated with 20mM Tris/HCl buffer (pH 7.4). Non-specifically bound proteins were firstremoved by washing with 20 mM Tris/HCl buffer (pH 7.4). rvWF was theneluted from the support by 270 mM NaCl in 20 mM Tris/HCl buffer (pH7.4). Subsequently, residual material was eluted from the column with 1MNaCl. During chromatography, the protein absorption was followed at 280nm in the customary manner. After chromatography, the proteinconcentration was determined according to the Bradford method (M.Bradford, loc.cit.). The content of rvWF was determined by means of acommercial ELISA system (Boehringer Mannheim).

It was found that nearly the total rvWF was bound to the support. Themajority of rvWF was eluted from the column in the elution with 270 mMNaCl, while the wash with 1M NaCl only contained traces of rvWF. Theresults of this purification step are summarized in Table 5. The portionof rvWF protein to total protein was increased to over 86% by thispurification step.

The fraction of 270 mM NaCl was more precisely examined with denaturingSDS protein gel electrophoresis (U. K. Laemmmli, Nature 227 (1970)680-685) and subsequent Western blot.

As presented in FIG. 5, the denaturing electrophoretic analysisestablished that rvWF was recovered at high purity by the purificationdescribed in Example 5 and Example 6. In the product isolated in thismanner, no other coagulation factors, such as Factor VIII, could bedetected.

                  TABLE 5    ______________________________________               volume   Total Protein                                  rvWF   rvWF/    Sample     (ml)     (μg/ml)                                  (μg/ml)                                         total protein    ______________________________________    rvWF concentrate               225      50        13.9   0.27    Elution with               43       70        60     0.86    270 mM NaCl    Elution with               32       25        2      0.08    1 M NaCl    ______________________________________

EXAMPLE 7

Isolation of plasmic and recombinant von Willebrand Factor withdifferent multimerization and characterization of the binding to FactorVIII

Recombinant von Willebrand Factor (r-vWF), von Willebrand Factor fromhuman plasma (p-vWF) and von Willebrand Factor from plasmacryoprecipitate (k-vWF) were purified by a combination of anion exchangechromatography and heparin affinity chromatography.

In anion exchange chromatography, the starting material (r-vWF:fermentation supernatant of recombinant CHO cells; p-vWF: human citrateplasma; k-vWF: plasma cryoprecipitate) was applied on aFraktogel-EMD-TMAE column (Merck) and vWF was obtained by elution with20 mM Tris/HCl buffer, pH 7.0, 280 mM NaCl. Subsequently, thepreparations were diluted to a salt concentration of 90 mM by additionof 20 mM Tris/HCl buffer and applied to a Fraktogel-EMD-heparin column(Merck). vWF was eluted by a stepwise elution with increasing saltconcentration. Thereby, the NaCl concentration in 20 mM Tris/HCl bufferwas varied between 120 mM and 280 mM. In this manner, preparations forp-vWF, k-vWF as well as r-vWF which differ from each other in thecomposition of multimers were obtained at 120 mM NaCl, 160 mM NaCl, 190mM NaCl, 230 mM NaCl and 280 mM NaCl.

The content of vWF (vWF-antigen, vWF:Ag) was determined by means of acustomary ELISA test (Asserachrom vWF, Boehringer Mannheim).

The binding of coagulation Factor VIII to p-vWF, k-vWF as well as r-vWFwas determined by means of "real-time biospecific interaction analysis"based on measurements from surface plasmon resonance technology(Malmqvist, M. Nature 1993; 361: 186-187). Thereby, the stochiometricratio of vWF subunit to bound Factor VIII was determined: a monoclonalanti-human von Willebrand antibody (AvW8/2) was covalently bound to thesensorchip CM5 (Pharmacia Biosensor AB) (O'Shannessy, D. J. et al., 1993Anal. Biochem. 212: 457-468; Karlsson, R. 1994 Anal. Biochem. 221:142-151). Thereby, the measurement of resonance units (RU) correspondsto the baseline (RU_(BL)).

p-vWF, k-vWF and r-vWF were dissolved to a concentration of 20 μg/ml in10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 0.05% surfactant P20 (HBSbuffer). For each experiment, a 50 μl sample of vWF was applied over thesensorchip with a flow speed of 5 μl/min in order to permit the bindingof AvW8/2 to vWF (phase A). Non-bound vWF is removed by washing with HBSbuffer (phase B). p-vWF and k-vWF were additionally washed with 20 μl250 mM CaCl₂ in order to remove traces of plasmic Factor VIII. Thebinding of vWF to AvW8/2 corresponds to the vWF resonance unit(RU_(vWF)). For the analysis of the binding of Factor VIII, 60 μl ofrecombinant FVIII (2.5 μg/ml in HBS buffer) was injected over thesensorchip at a constant flow rate of 5 μl/min in order to permit thebinding of Factor VIII to the vWF (phase C). The measurement of theresonance units corresponds to the FVIII binding (RU_(FVIII)). FactorVIII was dissociated again from vWF by washing with HBS buffer (phaseD). The stochiometric ratio of the complex of FVIII and vWF subunit isestablished from the resonance units and the molecular weights: vWF(subunit): FVIII=(RU_(vWF) -RU_(BL)) /(RU_(FVIII)-RU_(vWF))×330,000/220,000 (O'Shannessy, D. J. et al., 1993 Anal.Biochem. 212: 457-468; Karlsson, R. 1994 Anal. Biochem. 221: 142-151).FIG. 6 shows the sensogram of the attachment of r-Factor VIII to r-vWF.

                  TABLE 6    ______________________________________    Determination of the stochiometry of the vWF-Factor VIII complex                    Stochiometry    Sample          vWF subunit: FVIII    ______________________________________    r-vWF 120 mM eluate                    2.25:1    r-vWF 160 mM eluate                    2.5:1    r-vWF 190 mM eluate                    2.0:1    r-vWF 230 mM eluate                    2.0:1    r-vWF 280 mM eluate                    2.0:1    k-vWF 280 mM eluate                    2.6:1    p-vWF 280 mM eluate                      3:1    ______________________________________

Table 6 shows the stochiometry of vWF:Factor VIII of low and highmolecular fractions of plasmic and recombinant vWF. The data show thatthe low molecular as well as the high molecular fractions of rvWF have ahigher binding capacity to Factor VIII than p-vWF.

EXAMPLE 8

Purification and separation of plasmic vWF, vWF from cryoprecipitate andrecombinant vWF

Plasmic vWF (p-vWF), vWF from cryoprecipitate (k-vWF) and recombinantvWF (r-vWF) were purified by means of heparin affinity chromatographyand the low molecular and high molecular fractions were separatedaccording to Example 1.

From FIG. 7, it is recognizeable that the individual fractions of p-vWFand k-vWF have bands which are substantially less sharp than those ofr-vWF. This is even more clear in FIG. 8 where the fractionatedmultimers of p-vWF and r-vWF were directly compared. The sharp bands ofr-vWF clearly visible after separation in the SDS gel, whereasunambiguous intermediates with different molecular weights appear in thefractionation of p-vWF. These "intermediate bands" are traced back tothe heterogeneous mixture of differently sized vWF subnits in the p-vWFfractions which arise through digestion with with proteases present inproducts are known in the plasma. These intermediate products are knownin the literature as so-called triplet structures.

It is to be understood that the description, specific examples and data,while indicating preferred embodiments, are given by way of illustrationand exemplification and are not intended to limit the present invention.Various changes and modifications within the present invention willbecome apparent to the skilled artisan from the discussion anddisclosure contained herein.

We claim:
 1. A method for separating von Willebrand Factor (vWF) preparation into at least one fraction of high molecular weight vWF molecules having subunits with identical structure and at least one fraction of low molecular weight vWF molecules, comprisingcontacting the vWF preparation with a heparin affinity support to bind vWF to the heparin affinity support; and eluting vWF from the heparin affinity support with buffer having different salt concentrations, wherein the low molecular weight vWF molecules elute at a salt concentration between 150 mM and 250 mM and the high molecular weight vWF molecules elute at a salt concentration greater than 250 mM.
 2. The method according to claim 1, wherein the vWF preparation is obtained from a plasma fraction.
 3. The method according to claim 1, wherein the vWF preparation is obtained from cells transformed with vWF DNA.
 4. The method according to claim 1, wherein the buffer is free of Ca²⁺.
 5. The method according to claim 1, wherein the contacting is performed at a salt concentration that is less than 150 mM.
 6. The method according to claim 1, wherein low molecular weight vWF molecules are eluted at a salt concentration of about 160 mM.
 7. The method according to claim 1, wherein high molecular weight vWF molecules are eluted at a salt concentration greater than 270 mM.
 8. The method according to claim 1, wherein the salt is a monovalent salt or a divalent salt.
 9. The method according to claim 8, wherein the salt is sodium chloride.
 10. The method according to claim 1, wherein the heparin affinity support is selected from the group consisting of AF-HEPARIN TOYOPEARL®, HEPARIN EMD-FRAKTOGEL® and HEPARIN SEPHAROSE FAST FLOW®. 